Polypeptides comprise a wide variety of biological molecules, each having specific amino acid sequence, structure, and function. Most polypeptides interact with specific substances to carry out the function of the polypeptide. For instance, enzymes such as subtilisin or amylase interact with and hydrolyze specific substrates whereas proteinaceous cytokines or hormones typically interact with specific receptors to regulate, for example, growth or metabolism.
Efforts have been undertaken to alter characteristics or functional properties of various polypeptides by modifying the polypeptides' respective amino acid sequences. One approach has been to substitute one or more amino acids in the sequence of a polypeptide with a different amino acid(s) using in vitro mutagenesis techniques. As reported in the literature, such methods have been conducted to improve thermal or oxidative stability of various polypeptides. [See, e.g., Villafranca et al., Science, 222:782–788 (1983); Perry et al., Science, 226:555–557 (1984); Estell et al., J. Biol. Chem, 260:6518–6521 (1985); Rosenburg et al., Nature, 312:77–80 (1984); Courtney et al., Nature, 313:149–157 (1985)]. In addition, such methods have been reportedly used to generate enzymes with altered substrate specificities [See, e.g., Estell et al., Science, 223:655–663 (1986); Craik et al., Science, 228:291–297 (1985); Wells et al., Proc. Natl. Acad. Sci., 84:1219–1223 (1987)].
The structural biology of various enzymes has also been examined in the literature in an effort to better understand enzyme catalysis. For instance, studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases and glycosylases have been conducted by various researchers [Vihinen et al., J. Biochem., 107:267–272 (1990); Holm et al., Protein Engineering, 3:181–191 (1990); Takase et al., Biochemica et Biophysica Acta, 1120:281–288 (1992); Matsui et al., Febs Letters, 310:216–218 (1992); Matsui et al., Biochemistry, 33:451–458 (1992); Sogaard et al., J. Biol. Chem., 268:22480–22484 (1993); Sogaard et al., Carbohydrate Polymers, 21:137–146 (1993); Svensson, Plant Mol. Biol., 25:141–157 (1994); Svensson et al., J. Biotech., 29:1–37 (1993)].
Various members of the cellulase family of enzymes have also been examined by way of structural studies. Davies et al., Biochemistry, 37:1926–1932 (1998) describe the crystallography analysis of endoglucanase, Cel5A, from the alkalophilic Bacillus agaradherans. Davies et al. identified the structure of the catalytic core of this enzyme by multiple isomorphous replacement. The authors report that Cel5A performs catalysis via a double-displacement mechanism and that the Bronsted acid/base and enzymatic nucleophile in the catalytic core of Cel5A are residues Glu139 and Glu228, respectively. [See also, Davies et al., Biochemistry, 37:11707–11713 (1998)].
Additional enzymes which have been studied are the serine proteases and hen egg white lysozyme (“HEWL”). Analyses of various serine proteases have revealed that these enzymes contain a triad of the residues Asp-His-Ser in the active site [Matthews et al., Nature, 214:652–656 (1967); Blow et al., Nature, 221:337 (1969)] and tend to have pH optima in the neutral to alkaline range [Dodson et al., Trends Biochem. Sci., 23:347 (1998)]. This type of triad has been observed in a number of diverse enzymes. Variations within such triads, however, have been described that catalyse the hydrolysis of many classes of substrates [Dodson et al., supra].
In the serine proteases, the triad in the active site can act as a charge-relay system [Blow et al., supra], wherein the histidine residue removes a proton from the serine residue to make it a more potent nucleophile. In this catalytic scheme, the formation of an unusually short catalytic hydrogen bond between the histidine and aspartate appears to be critical so as to make the histidine a more potent base by facilitating its deprotonation of serine. [Wang et al., J. Biol. Chem., 268:14096–14102 (1993)] This hydrogen has recently been visualized in an ultra-high resolution x-ray study of subtilisin [Kuhn et al., Biochemistry, 37:13446 (1998)].
HEWL contains two catalytic carboxylates, aspartate and glutamate, in the active-site [see, e.g., Blake et al., Nature, 206:757 (1965); Johnson et al., Nature, 206:761 (1965); Phillips, Harvey Lectures, 66:135 (1971); Ford et al., J. Mol. Biol., 88:349 (1974); Kelly et al., Nature:282:875 (1979)]. The glutamate residue in that active-site acts as an acid/base catalyst, initially protonating the glycosidic bond and catalysing bond fission. The aspartate residue in that active-site facilitates the reaction by stabilizing the resulting carbonium ion intermediate.
Similar to HEWL, various other enzymes have been reported to contain two carboxylates for catalysis, including certain acid proteases like the pepsin family [see, Hsu et al., Nature, 266:140–145 (1977)], certain retro-viral proteases [see, Miller et al., Nature, 337:576 (1989)], and the family of glucosyl hydrolases [see, Davies et al., Structure, 3:853 (1995); White et al., Curr. Op. Struct. Biol., 7:645 (1997)]. The pKa of a glutamate side-chain in solution is approximately 4.5, and as the acid/base group has to be protonated in the resting state, most of these types of enzymes tend to perform or have activity in acidic environments [White et al., supra]. However, some of these types of enzymes which utilize a dicarboxylate catalysis mechanism have pH optima in the neutral to alkaline range. To date, it has not been fully understood how such enzymes may accomplish such an increase in the pKa of the acid/base carboxylate group.